Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

Introduction

The cation chloride cotransporter family (CCCs) consists of electroneutral secondary active cotransporters that mediate the symport of cations (Na+ and K+) coupled with chloride ions (Cl-) across the plasma membrane. CCCs are divided into K+-Cl- outward cotransporters (KCC1-4), Na+, K+, Cl- inward cotransporters (NKCC1-2, NCCs), the polyamine transporter CCC9, and the CCC-interacting protein CIP1 [1-3]. These transporters play a crucial role in various physiological processes like regulation of cell volume, directional ion transport across epithelial cells, secretion of K+, and regulation of intracellular Cl−concentration ([Cl-]i) in neurons [2, 4–11].

In neurons, KCC2 and NKCC1 are involved in the development and maintenance of inhibitory neurotransmission. For instance, KCC2 generates a low [Cl-]i required for fast inhibitory neurotransmission. Binding of γ-aminobutyric acid (GABA) or glycine to their receptors, which are ligand-gated Cl- channels, leads to a Cl- influx and therefore hyperpolarization [8, 12]. By contrast, NKCC1 is more prevalent in immature inhibitory neurons. There, its action results in a high [Cl-]i causing GABA or glycine to elicit a depolarizing action that opens L-type voltage-gated Ca2+ channels necessary for proper synapse formation [13-18].

The physiological relevance of the two cotransporters is corroborated by the phenotypes present in knockout mice. Mice with disruption of the gene Slc12a5 encoding both KCC2 isoforms (KCC2a and KCC2b), die shortly after birth due to motor deficits [19, 20]. Furthermore, dysfunction of KCC2 is associated with neurological and psychiatric disorders including epilepsy, neuropathic pain, spasticity, ischemic insults, brain trauma, schizophrenia, and autism [21-32]. Mice with disruption of Slc12a2 (NKCC1) are viable, but suffer from peripheral deafness, postnatal hyperexcitability and anti-convulsant, pain perception and male infertility [33-36]. Inhibition of KCC2 promotes formation of pathological conditions whereas inhibition of NKCC1 seems to prevent or at least alleviate pathological phenotypes [37]. This renders these two transporters a prime pharmacotherapeutic target and fosters interest in understanding posttranslational mechanisms of their regulation [12, 38–40]. The main focus is set on phospho-related mechanisms. The broad kinase inhibitor staurosporine was used to enhance KCC2 transport activity in immature auditory brainstem [41] and hippocampal cultured neurons [42], and to decrease NKCC1 activity [43-46]. Furthermore, N-ethylmaleimide (NEM), that likely modulates the activity of kinases and phosphatases, activates KCC2 [47-50] and inhibits NKCC1 [45, 51]. Thus, both compounds reciprocally regulate KCC2 (activation) and NKCC1 (inhibition). Understanding their mode of action provides the basis for further pharmacological studies and important insight into the molecular mechanisms involved in physiological regulation of these two transporters.

Since KCC2 and NKCC1 are present in the same neuronal populations but transport K+ and Cl- in opposite direction [52-54], regulatory mechanisms are required to coordinate their activity [4, 38, 55–60]. Indeed, several kinases and phosphatases regulate their transport activity. In vitro and in vivo analyses revealed that members of the with-no-lysine kinase (WNKs) family in combination with their downstream targets STE20/SPS1-related proline/alanine rich kinase (SPAK) and oxidative stress response kinase (OSR1) are the most prominent kinases that regulate KCC2 and NKCC1 activity in a reciprocal way. SPAK/OSR1, activated by WNK1, phosphorylate Thr6 and Thr1007 of KCC2 [48, 59, 61–64]. WNKs also interact with a yet unknown kinase to phosphorylate Thr906 of KCC2 [48, 63]. Site-directed mutagenesis studies indicate that simultaneous mutation of Thr906 and Thr1007 to alanine (mimicking the dephosphorylated state) activates KCC2 [61, 65–67]. WNK/SPAK/OSR1 also phosphorylates the N-terminal residues Thr203, Thr207, Thr212 and Thr217 of NKCC1 [38, 59, 60, 68–76] thereby activating NKCC1 [72, 75]. Thus dephosphorylation (KCC2) and phosphorylation (NKCC1) of WNK/SPAK/OSR1 specific phospho-sites enhance KCC2 and NKCC1 activity.

Phosphorylation can also enhance KCC2 activity. Phosphorylation of Ser932, Thr934, Ser937, or the protein kinase C (PKC) site Ser940, all residing in exon 22, increases its transport activity [47, 68, 77]. The multiplicity of phosphorylation/dephosphorylation sites on KCC2 offers a complex toolbox to gradually fine-regulate its activity and integrate different signaling pathways [4, 40, 65, 68].

Initially, both staurosporine and NEM were thought to act through a similar mechanism [47], but recent findings revealed that they act differentially on specific KCC2 phospho-sites [40]. Furthermore, staurosporine and NEM mediated effects involve both KCC2 phosphorylation and dephosphorylation [40, 48]. To gain insight into their mode of action, we analyzed the impact of these compounds on phosphorylation of specific KCC2 and NKCC1 phospho-sites using large-scale phosphoproteomics studies and phospho-site specific antibodies in stably transfected HEK293 cells and immature primary cultures of hippocampal neurons.

Material and methods

Cell culturing of HEK293 cells

For K+-Cl- cotransporter activity measurements, stably transfected rat KCC2b HEK293 cells (HEK) [55] were plated in a 0.1 mg/ml poly-L-lysine coated black-well 96 well culture dish (Greiner Bio-One, Frickenhausen, Germany) at a concentration of 100,000 cells/well. The remaining cells were plated on a 0.1 mg/ml poly-L-lysine-coated class coverslip and ~18 h later analyzed by immunocytochemistry.

Primary cultures of rat hippocampal neuron

All animal procedures were carried out in accordance with the European Union Directive of 22 September (2010/63/EU). The protocol was approved by the INSERM Local committee (Number 0287.01, delivered by Ministère de l’Education et de la Recherche). Hippocampi from 18-day-old rat embryos were dissected and dissociated using trypsin (0.05%) and plated in 60-mm culture dishes at a density of 100,000 cells cm−2 in minimal essential medium (MEM) supplemented with 10% NU serum (BD Biosciences, Le Pont de Claix, France), 10 mM glucose, 1 mM sodium pyruvate, 2 mM glutamine, and 100 U ml−1 penicillin–streptomycin as previously described [78]. On day 7 of culture incubation, half of the medium was changed to MEM with 2% B27 supplement (Invitrogen). 24 h prior to plating, dishes were coated with poly-ethylenimine (5 μg/ml).

Immunocytochemistry

For immunocytochemistry, all steps were performed at room temperature. HEK293 cells grown on poly-L-lysine-coated coverslips were fixated for 10 min with 4% paraformaldehyde in 0.2 M phosphate buffer (PBS). After three washing steps in PBS, cells were incubated with blocking solution (2% bovine serum albumin, 10% goat serum in PBS) for 30 min. Primary antibody solution (anti-KCC2 N1-12; 1:1,000; Neuromab, Carlifornia, USA) was added in carrier solution (0.3% Triton X-100, 1% bovine serum albumin, 1% goat serum in PBS) for 1 h. After washing three times in PBS, the secondary antibody, which was conjugated to a fluorescent probe (Alexa Fluor 488 goat anti-mouse; 1:1,000; Thermo Fisher Scientific, Bremen, Germany) was incubated for 1 h. After three washes in PBS, cells were mounted onto glass slides with Mowiol (Roth, Karlsruhe, Germany) and DAPI (Roth; 1:1,000). Photomicrographs were taken using Biozero BZ-8100E (Keyence, Neu-Isenburg Deutschland) fluorescence microscope (Leica, Wetzlar, Germany).

Determination of K+-Cl- cotransport

KCC2 transport activity was determined by Cl−dependent uptake of Tl+ in HEK293 cells as previously described [47, 79, 80]. To initiate the flux measurement, the medium in the 96-well culture dish was replaced by 80 μl preincubation buffer (100 mM N-methyl-D-glucamine-chloride, 5 mM HEPES, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, pH 7.4, 160 mmol / kg ± 2,04 mmol/kg) supplemented with 2 μM FlouZin-2 AM dye (Thermo Scientific, Bremen, Germany) plus 0.2% (wt/vol) Pluronic F-127 (Thermo Scientific, Bremen, Germany) and incubated for 48 min at room temperature. Cells were then washed 3 times with 80 μl preincubation buffer and incubated for 15 min with 80 μl preincubation buffer plus 0.1 mM ouabain to block Na+/K+ ATPase activity. Three technical and five biological replicates were performed for each construct. Afterwards, the 96-well plate was placed into a fluorometer (Fluoroskan Accent, Thermo Scientific, Bremen, Germany) and each well was injected with 40 μl 5 x Tl+ stimulation buffer (12 mM Tl2SO4, 100 mM NMDG, 5 mM Hepes, 2 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, pH 7.4; 175 mmol/kg ± 2 mmol/kg). Fluorescence was determined in a kinetic dependent manner (excitation 485 nm, emission 538 nm, 1 frame in 5 s in a 200 s time span) across the entire cell population in a single well. By using linear regression of the initial values of the slope of Tl+-stimulated fluorescence increase, transport activity was calculated [79, 80].

To determine the dose-response profile, increasing concentration of staurosporine (8–80 μM) and NEM (25–3000 μM) were applied to the preincubation buffer 15 min prior flux measurement. This was done for three technical replicates and at least five independent measurements were performed.

Statistical analyses

The majority of data populations illustrated in Figs 1, 3 and 4 showed non-normal distributions (verified using Shapiro-Wilk normality test at 0.05 significance level). Therefore, a nonparametric test (Wilcoxon-Mann-Whitney rank sum test) was employed for the comparison between different groups of data. The resulting p-values were adjusted with the Bonferroni correction for multiple testing. Note that only p-values < 0.01 were considered to reduce the chances of false positives (type I errors). The sample size n always refers to the number of biological replicates (independent preparations). The activity of each independent preparation was determined as mean over three technical replicates. The average scatter within technical replicates was 3 times smaller than the scatter across biological replicates for a given treatment.

Fig 1

Staurosporine and NEM increase KCC2 activity.

A: Immunoreactivity of stably transfected HEK cells was detected with labeling at the cell membrane and cytosol. Photomicrographs were taken with a confocal laser-scanning microscope. Scale bar, 10 μm. B: Transport activity of rnKCC2b was determined by Tl+ flux measurements. Treatment with 10 μM staurosporine or 1 mM NEM resulted in an approximately twofold increase of rnKCC2b activity. Application of the loop diuretics furosemide (2 mM) significantly inhibited rnKCC2b function. The graph represents the data of at least five independent measurements (each consisting of three technical replicates) normalized to rnKCC2b. Statistical analyses: ***, p < 0.001 versus HEK; Error bars represent SD.

Fig 3

Quantitative analyses of rnKCC2b and hsNKCC1 phospho-sites upon staurosporine and NEM treatment in HEKrnKCC2b cells.

Stably transfected HEK cells were treated with DMSO (control), 8 μM staurosporine or 0.5 mM NEM, respectively, for 15 min. Cell lysates were harvested and subjected to immunoprecipitation (IP) and immunoblot (IB) with indicated antibodies. D, dimeric KCC2; M, monomeric KCC2. Band intensities were quantified with ImageJ software. ***, p < 0.001; **, p < 0.01; Wilcoxon-Mann-Whitney test (n = 6).

Fig 4

Quantitative analyses of rnKCC2 and rnNKCC1 phospho-sites upon staurosporine or NEM treatment in immature hippocampal neurons.

Immature rat hippocampal neurons were treated with DMSO (control), 8 μM staurosporine or 0.5 mM NEM, respectively, for 15 min. Neuronal lysates were harvested and subjected to immunoprecipitation (IP) and immunoblot (IB) with the indicated antibodies. D, dimeric KCC2; M, monomeric KCC2. Band intensities were quantified using ImageJ software. ***, p < 0.001; **, p < 0.01; Wilcoxon-Mann-Whitney test (n = 6).

For dose-response analyses, we used the nls function from the stats package in R (version 3.5.1) to model the data points with the Hill-Langmuir equation, where y is the Tl+ uptake of HEK cells, y0 is the Tl+ independent-baseline activity (y0 = 100%), ysat is the maximum activity (equivalent to Vmax in reaction rate equation) relative to baseline, x is the concentration of the agonist, n is the Hill coefficient, and ED50 is the agonist concentration that produces 50% of the saturation response. The 95% confidence intervals for the dose response curves were determined with the function NonlinearModelFit [“MeanPredictionBands”] in Wolfram Mathematica. For the boxplots, the box extends from the upper (Q1) to the lower (Q3) quartile. The line inside the box represents the median. The whiskers extend to the outermost data point that falls within upper inner and lower inner Quartile fences [Q1+1.5(IQR)] and [Q3-1.5(IQR)], respectively, where IQR = Q1-Q3 is the interquartile range.

Treatment of stably transfected HEKrnKCC2b cells and hippocampal neurons

For mass spectrometry and immunoblot analyses, stably transfected HEK cells [55] were grown in 75 cm² cell-culture flask. Cells were washed with 5 ml flux hypotonic preincubation buffer (100 mM N-methyl-D-glucamine-chloride, 5 mM HEPES, 5 mM KCl, 2 mM CaCl2, 0.8 mM MgSO4, 5 mM glucose, pH 7.4; 160 mmol/kg±2,04 mmol/kg) and then treated with or without 8 μM staurosporine or 1 mM NEM. Cells were centrifuged at 500 rpm for 3 min and the resulting pellets used for immunoblot analyses or mass spectrometry analyses.

For immunoblot analyses of immature (9 days in vitro, (DIV)) rat hippocampal neurons, half of the media from culture dishes containing neurons (2.5 ml from each dish) was collected to prepare samples including 16 μM staurosporine (from 10 mM stock solution in DMSO), 1 mM NEM (by direct dissolving of the NEM powder) or DMSO (vehicle, same volume as for staurosporine), respectively. The samples were distributed dropwise into the culture dishes that gave final concentration of 8 μM staurosporine or 0.5 mM NEM and cultures were incubated 15 min at 37 °C (5% CO2). After incubation, neurons were rinsed twice with Hanks’ Balanced Salt solution (HBSS, ThermoFisher Scientific) precooled to 4 °C. The rinsage solution was replaced with ice-cold HBSS containing a cocktail of protease and phosphatase inhibitors (ThermoFisher Scientific) and neurons rapidly scraped and centrifuged at 4 °C (10,000 g, 3 min). Pellets were frozen in liquid nitrogen and kept for future analyses at -80 °C.

Multi-Enzyme Digestion Filter Aided Sample preparation (MED FAS)

For mass spectrometry analyses, cell pellets of untreated, staurosporine or NEM treated HEK cells were lysed in 2% SDS in 0.1 M Tris-HCl, pH 8.0, containing 0.1 M DTT as described previously [81]. Total protein was determined using WF-assay in micro-titer plate format [82]. Aliquots of the cell lysate containing 1–2 mg were processed in Amicon Ultra 15 Ultracel 30k (Merck Millipore, Darmstadt) devices as described previously [83] with several modifications using the MED FASP method [84]. Briefly, SDS and other low molecular weight material were removed by centrifugation at 4,000 x g in 5 consecutive washes with UA buffer containing 8 M urea (ultrapure, Merck, Darmstadt) in 0.1 M Tris pH 8. Following two washes with 5 ml of 0.1M Tris-HCl, pH 8 (DB buffer), 20 μg of LysC (Wako, Neuss) in 0.5 mL of DB was added to the filter. Samples were digested overnight at 37 °C and peptides were collected by centrifugation. Next, the material retained in the filter was cleaved with 10 μg trypsin in 0.5 ml DB at 37 °C for 4 h and the peptides were eluted as previously. To increase the yield of peptides, filters were washed twice with 0.5 mL DB. Concentration of peptides was determined by WF-assay [82].

TiO2-based enrichment of phosphopeptides

Phosphopeptides were enriched using TiO2-beads [85] with several modifications [83]. Briefly, 25 mg of ‘Titansphere TiO2 10 μm’ (GL Sciences, Inc., Japan) were suspended in 50 μl of 3% (m/v) dihydroxybenzoic acid in 80% (v/v) CH3CN, 0.1% CF3COOH and diluted 1:4 with water before use. Ten microliters of this slurry (1 mg beads) were added and samples incubated under continuous agitation for 20 min. The mass ratio of the beads and peptides was 3:1. Then, the titanium beads were sedimented by centrifugation at 5,000 x g for 1 min and the supernatants were collected and mixed with another portion of the beads and incubated as above. The bead-pellets were resuspended in 150 μl of 30% (v/v) CH3CN containing 3% (v/v) CF3COOH and transferred to a 200 μl pipet tip plugged with one layer of glass microfiber filter GFA (Whatman). The beads were washed three times with 30% (v/v) CH3CN, 3% CF3COOH (v/v) solution and three times with 80% CH3CN (v/v), 0.3% CF3COOH (v/v) solution. Finally, the peptides were eluted from the beads with 100 μL of 40% CH3CN (v/v) containing 15% NH4OH (m/v) and were vacuum-concentrated to ∼4 μl.

Mass-spectrometric analysis

Analysis of peptide mixtures was conducted using a QExactive HF-X mass spectrometer (Thermo-Fisher Scientific, Palo Alto). Aliquots containing <1 μg of total peptide were chromatographed on a 50 cm column with 75 μm inner diameter packed C18 material. Peptide separation occurred at 300 nl/min for 95 min using an acetonitrile gradient of 5–30%. The temperature of the column oven was 55°C. The mass spectrometer operated in data-dependent mode with survey scans acquired at a resolution of 60,000. Up to the top 15 most abundant isotope patterns with charge ≥ +2 from the survey scan (300–1650 m/z) were selected with an isolation window of 1.4 m/z and fragmented by HCD with normalized collision energies of 25. The maximum ion injection times for the survey scan and the MS/MS scans were 20 and 28 ms, respectively. The ion target value for MS1 and MS2 scan modes was set to 3×106 and 105, respectively. The dynamic exclusion was 30 s. The MS spectra were searched using MaxQuant software. A maximum of two missed cleavages was allowed. The maximum false peptide and protein discovery rate was specified as 0.01. The whole mass spectrometry analyses were performed in three technical and two biological replicas per treatment.

Immunoblot and phospho-antibody immunoprecipitation analyses

Lysate protein samples were subjected to immunoblot and immunoprecipitation as previously described [63]. Protein samples (40 μg) were boiled in sample buffer for 5 min, resolved by 7.5% sodium dodecyl sulfate polyacrylamide-gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. Membranes were incubated for 30 min with TBST (Tris-buffered saline, 0.05% Tween-20) containing 5% (w/v) skim milk. Blots were then washed six times with TBST and incubated for 1 h at room temperature with secondary HRP-conjugated antibodies diluted 5000-fold in 5% (w/v) skim milk in TBST. After repeating the washing steps, signals were detected with enhanced chemiluminescence reagent. Immunoblots were developed using ChemiDoc™ Imaging Systems (Bio-Rad, Feldkirchen). Figures were generated using Photoshop/Illustrator (Adobe). Band densities of bands were measured with ImageJ. Calculation of intensity ratios is based on (phospho-dimeric KCC2 + phospho-monomeric KCC2) / (total dimeric KCC2 + total monomeric KCC2), (total dimeric KCC2 + total monomeric KCC2)/β-actin or tubulin, SPAK pThr233/SPAK, SPAK pSer373/SPAK, SPAK/β-actin or tubulin, NKCC1 pThr203,207,212/NKCC1, NKCC1/β-actin or tubulin, as reported previously [66]. Mann–Whitney U-test was used for comparison between 2 independent groups and Wilcoxon matched pairs test was employed to compare paired data (GraphPad Prism 7.0, San Diego, CA, USA).

For phospho-antibody immunoprecipitation, KCC isoforms were immunoprecipitated from indicated cell extracts. 2 mg of the indicated clarified cell extract was mixed with 15 μg of the indicated phospho-specific KCC antibody conjugated to 15 μl of protein-G–Sepharose, in the added presence of 20 μg of the dephosphorylated form of the phosphopeptide antigen, and incubated 2 hours at 4 °C with gentle shaking. Immunoprecipitates were washed three times with 1 ml of lysis buffer containing 0.15 M NaCl and twice with 1 ml of buffer A. Bound proteins were eluted with 1x LDS sample buffer.

Antibodies

The following antibodies were purchased from Division of Signal Transduction Therapy Unit at the University of Dundee: KCC2A phospho-Thr906 (SAYTYER(T)LMMEQRSRR [residues 975–989 of human KCC3A] corresponding to SAYTYEK(T)LVMEQRSQI [residues 899–915 of human KCC2A], S959C); KCC2A phospho-Thr1007 (CYQEKVHM(T)WTKDKYM [residues 1032–1046 of human KCC3A] corresponding to TDPEKVHL(T)WTKDKSVA [residues 998–1014 of human KCC2A], S961C); SPAK/OSR1 (T-loop) phospho-Thr233/Thr185 antibody (226–238 of human SPAK or residues 178–190 of human OSR1, TRNKVRKpTFVGTP, S204C); SPAK/OSR1 (S-motif) phospho-Ser373/Ser325 antibody (367–379 of human SPAK, RRVPGSpSGHLHKT, which is highly similar to residues 319–331 of human OSR1 in which the sequence is RRVPGSpSGRLHKT, S670B); SPAK (427–443 of human SPAK, QSLSVHDSQGPPVGTP, S849C); NKCC1 phospho-Thr203+Thr207+Thr212 (residues 198–217 of human NKCC1, HYYYDpTHTNpTYYLRpTFGHNT, S763B); NKCC1 phospho-Thr212/Thr217 (residues 208–223 of human NKCC1, YYLRpTFGHNpTMDAVPR, S063D); NKCC1-total antibody (residues 1–260 of shark NKCC1, S841B). Pan-KCC2 antibody (residues 932–1043 of human KCC2) was from NeuroMab (73–013). KCC2 phospho-Ser940 antibody was from ThermoFisher Scientific (PA5-95678). PKCδ phospho-Thr505 antibody (9374) and β-Actin (D6A8) antibody (12620) were from Cell Signaling Technology. Anti (neuronal)-β-Tubulin III antibody was from Sigma-Aldrich (T8578). Secondary antibodies coupled to horseradish peroxidase used for immunoblotting were obtained from Pierce. IgG used in control immunoprecipitation experiments was affinity-purified from pre-immune serum using Protein G-Sepharose.

Results

Dose-response analyses of staurosporine and NEM in stably transfected HEKrnKCC2b cells

Staurosporine and NEM generally activate KCCs [40, 47, 86, 87]. To closer characterize their mode of action, we used stably transfected HEK cells, as high amount of the cotransporter is advantageous for subsequent biochemical analyses. Immunoreactivity of HEK cells against KCC2 was detected in all cells with clear labeling at the cell membrane and cytosol (Fig 1A). This is in agreement with previous cell surface expression analyses that detected 11.8 ± 1.4% of total KCC2 in stably transfected rnKCC2b at the cell surface [79].

To quantify the impact of staurosporine and NEM on rnKCC2b transport activity, Tl+ flux measurements were performed [79, 80, 88]. Transport activity in HEK cells was 5.6 times higher compared to background (HEK 100% ± 5.32%; mock; 17.7% ± 5.5%; p = 4.3x10-5; n = 5) (Fig 1B). Here and elsewhere in the text, numbers indicate mean +/- SD, p was determined using Wilcoxon-Mann-Whitney rank sum test. Treatment with 10 μM staurosporine or 1 mM NEM resulted in an approximately twofold increase of rnKCC2b activity compared to untreated HEK cells (staurosporine: 187.5% ± 37.1%, p = 3x10-3; NEM: 204% ± 23.2%; p = 3x10-3). The loop diuretics furosemide, that specifically inhibits the function of cation chloride cotransporters [89, 90], significantly inhibited the function of rnKCC2b (34.7 ± 12.2%, p = 4 x10-5).

Next, we determined the dose-response relationships of staurosporine and NEM on rnKCC2b transport activity by treating HEK cells with different concentrations of staurosporine (5–80 μM) or NEM (25–3,000 μM). Fig 2 represents the dose-response curve for both agents. The dose-response curve for staurosporine (Fig 2A) was approximately a rectangular hyperbola (n ≈ 1, not significantly different from unity). This reflects Michaelis-Menten kinetics and suggests absence of cooperative effects. In contrast, the dose response curve for NEM (Fig 2B) had a pronounced sigmoidal shape (n ≈ 5), which reflects cooperative binding kinetics. The effective dose ED50 (representing the potency) for staurosporine of stably transfected HEK was 12.8 ± 4.9 μM and the maximal efficacy (Emax) was 205 ± 40%. The ED50 value for NEM was 0.5 ± 1.3 mM and Emax was 105 ± 6%. For further analyses, we used a concentration of 8 μM staurosporine and 1 mM NEM, if not indicated otherwise as these concentrations significantly increase KCC2b transport activity.

Fig 2

Dose-response relationship of staurosporine and NEM on KCC2 transport activity.

HEK cells were treated with different concentrations of staurosporine (5–80 μM; A) or NEM (25–3,000 μM; B) for 15 min. Transport activity of rnKCC2b was determined by Tl+ flux measurements. Treatment with staurosporine resulted in a classical Michaelis-Menden curve, whereas NEM treatment showed a sigmoidal curve with a Hill coefficient of 5.2 ± 1.3 indicating a positive cooperative effect on rnKCC2b. The graph represents the data of at least five independent measurements (each consisting of three technical replicates) normalized to untreated HEK.

Identification of CCC phosphorylation sites in stably transfected HEKrnKCC2b cells by mass spectrometry analyses upon treatment with staurosporine or NEM

Phosphoproteomics by mass spectrometry has the advantage of providing an unbiased survey of phospho-sites. Therefore, we here used for the first time this technique to gain insight on the impact of staurosporine and NEM on rnKCC2b phospho-sites in HEK. For each condition, two biological and three technical replicas were performed. Phospho-sites detected in at least one technical experiment are listed in Table 1. We first mapped KCC2b phospho-sites in untreated HEK cells. Twelve phospho-sites were identified: Ser25, Ser26, Ser31, Thr32, Thr34 in the cytoplasmic N-terminus and Thr906, Ser937, Ser940, Thr1007, Ser1022, Ser1025, and Ser1026 in the C-terminus (Table 1, untreated). These sites include all phospho-sites already present in PhosphositePlus (Table 2) [91]. Recently phosphoproteomic data deposited in Phosida and Phosphosite plus revealed that rat KCC2b tissue only harbors seven phospho-sites (Table 2). Thus, we here report five phospho-sites (Ser25, Thr32, Thr906, Ser937 and Thr1007) that were so far only reported for mouse but not rat KCC2 tissue (Table 2). These sites most likely reflect different expression of kinases and phosphatases in different tissues (HEK293 vs. rat brain tissue) [69] or increased detection rate in stably transfected HEK cells.

Table 1

Phospho-sites of stably transfected HEKrnKCC2b cells.

Stably transfected HEK were treated with or without 1 mM NEM or 8 μM staurosporine before they were analyzed by mass spectrometry. The protein accession numbers are: hsNKCC1 (P55011), hsKCC1 (Q9UP95), rnKCC2b (Q9H2X9-2), hsKCC4 (Q9Y666).

Sample	Phosphorylation sites	Phosphopeptides	untreated	NEM treated	staurosporine treated
hsNKCC1	T212; T217	TFGHNTMDAVPR	(2)
	S242	LLRPSLAELHDELEK	(1)	(1)
	T266	EPFEDGFANGEESTPTR	(3)	(2)	(2)
	S940	EGLDISHLQGQEELLSSQEK	(2) (2)	(2) (1)	(3) (1)
	Y956; S957	DVVVSVEYSKK	(3) (2)	(3) (1)	(3) (3)
hsKCC1	T983	IQMTWTRDK	(2)
rnKCC2b	S25; S26	ESSPFINSTDTEK	(1)
	S26	ESSPFINSTDTEK	(2) (3)	(3) (3)	(3) (3)
	S31	ESSPFINSTDTEK	(2)	(1)(1)
	T32	ESSPFINSTDTEK	(1)	(1)	(1)
	T34	ESSPFINSTDTEK	(3) (2)	(2)	(1)
	T906	TLVMEQRSQILK	(3)	(3)	(3)
	S937	EIQSITDESRGSIR	(2)
	S940	EIQSITDESRGSIR	(2) (3)	(2)	(1)
	T1007	VHLTWTK	(3)	(3)	(3)
	S1022; S1025	GPSPVSSEGIK	(3) (1)	(3)	(3) (1)
	S1022; S1025; S1026	GPSPVSSEGIK	(1)	(1)
	S1022; S1026	GPSPVSSEGIK	(3)	(3)	(3)
	S1026	GPSPVSSEGIK	(3)	(3)	(3)
hsKCC4	T926	TLMMEQR	(2)
	T980	VQMTWTREK	(3)	(1)

The number in brackets indicates in how many technical replica a given phospho-site was detected (max. 3). Each bracket provides the results of one biological experiment.

Table 2

Phospho-sites in PhosphoSitePlus and PHOSIDA detected by mass spectrometry analyses.

Phosphosite Plus rnKCC2b	Phosphosite Plus hsKCC2b	Phosphosite Plus mmKCC2b	Phosida mmKCC2b	Transport activity measured by:
-	-	S25	S25	[47, 65]
S26	-	S26	S26	[47, 65]
S31	S31	-	S31	(40)
-	-	-	(T32)	-
T34	T34	T34	T34	[40, 92]
-	Y594	-	-	-
-	S599	-	-	-
-	-	-	S649	-
-	T798	-	-	-
-	T808	-	-	-
-	-	T906	T906	[47, 61, 66]
-	-	S913	-	-
-	-	S932	(S932)	[40]
-	-	-	(T934)	[47]
-	-	S937	S937	[47]
S940	-	S940	S940	[48, 77]
-	-	S988	-	-
-	-	T999	-	[40]
-	-	T1006	T1006	[47, 48, 61, 66]
-	-	-	T1008	[40]
S1022	-	S1021	S1021	[47]
S1025	-	S1024	(S1024)	[47]
S1026	-	S1025	S1025	[47]
-	-	S1044	-	-

Abbreviations used are as follows: rn, Rattus norvegicus; hs, Homo sapiens; mm, Mus musculus. Phospho-sites detected in the present mass spectrometry study are marked in bold.

As KCC1, KCC4, and NKCC1 are endogenously expressed in HEK293 cells and as mass spectrometric analysis provides data on most proteins in a given sample, we also investigated phosphorylation sites of the following proteins: hsKCC1 (Thr893, analogous to rnKCC2b Thr1007), hsKCC4 (Thr926 and Thr980, analogous to rnKCC2b Thr906 and Thr1007) and hsNKCC1 (Thr212, Thr217, Ser242, Thr266, Thr268, Ser940, Tyr956, and Ser957). These phospho-sites were already previously deposited in PhosphositePlus and Phosida (S1–S3 Tables). Overall, we detected only a low proportion of all so far deposited phospho-sites for these three cotransporters. This might reflect low expression levels in HEK293 cells.

Next, we investigated the phosphorylation pattern of rnKCC2b upon staurosporine and NEM treatment. Phosphorylation at Ser26, Thr32, Thr34, Thr906, Ser940, Thr1007, Ser1022, Ser1025, Ser1026 of rnKCC2b were still present upon treatment with staurosporine or NEM, whereas phosphorylation of Ser25 and Ser937 was not detected anymore in either of the two conditions (Table 1). Additionally, no phosphorylation was detected for Ser31 after treatment with staurosporine.

Regarding endogenously expressed CCCs, the phospho-site Thr983 of hsKCC1 (analogous to rnKCC2b Thr1007), Thr926 of hsKCC4 (analogous to rnKCC2b Thr906) and Thr212/217 and Thr266/268 of hsNKCC1 could not be detected upon application of either of the two reagents. Additionally, the phospho-sites Thr980 of hsKCC4 (analogous to rnKCC2b Thr906) and Ser242 of hsNKCC1 were not detected anymore after staurosporine treatment. The phospho-sites Thr266, Ser940 and Tyr956/Ser957 of hsNKCC1 were still present upon treatment with staurosporine and NEM.

Several kinases were described to directly phosphorylate KCC2 and NKCC1. This includes kinases of the WNK-SPAK/OSR1 and PKC mediated phosphorylation pathways. To gain further insight into the regulatory phosphorylation mechanism, we explored their phosphorylation pattern as well. We detected several phosphorylation sites in hsWNK1, hsWNK2, and hsSPAK [69, 93, 94] (Table 3). Upon all, we observed phosphorylation of the activating T-loop residue Ser382 of hsWNK1 and the S-loop phosphorylation site of Ser372/373 of hsSPAK that is phosphorylated by WNK1 [63, 69, 93]. Upon treatment with staurosporine or NEM, Ser382 of hsWNK1 and Ser372/373 of hsSPAK were not detected anymore (Table 4). We were not able to detect phosphorylation of Thr233 that is located in the T-loop kinase domain of hsSPAK. Normally, this site is directly phosphorylated by WNK1 and WNK4 to activate SPAK [60, 63, 69, 95]. Furthermore, we did not detect any phospho-sites of the ubiquitously expressed WNK3 and WNK4. Again, this probably results from low expression rates in HEK293 cells.

Table 3

Phospho-sites of hsSPAK, hsWNK1 and hsWNK2 endogenously expressed in stably transfected HEKrnKCC2b cells.

Stably transfected HEK were treated with or without 1 mM NEM or 8 μM staurosporine before they were analyzed by mass spectrometry. The protein accession numbers are: hsSPAK (AAC72238.1), hsWNK1 (Q9H4A3.2), hsWNK2 (Q9Y3S1.4).

sample	Phosphorylation site	Phosphopeptides	untreated	staurosporine treated	NEM treated
hsSPAK	308	EMMKKYGK	(2) (1)	(1)	(2)
	354	LLTRTPDIAQRAK	(1)	(2)	(1)
	356	LLTRTPDIAQRAK	(2)	(2)	(1)
	403	AAFSQEK	(3)	(1)	(1)
	518; 520	ALKTLTFK	(3) (2)	(2) (3)	(3) (3)
	372; 373	VRRVPGSSGHLHK	(1)
hsWNK1	11	QSSTPGSLFLSPPAPAPK	(2)	(1)
	167; 170	DRPVSQPSLVGSK	(3)	(2)	(3)
	167; 174	DRPVSQPSLVGSK	(3)	(3)	(2)
	170	DRPVSQPSLVGSK	(3)		(2)
	174	DRPVSQPSLVGSK	(3) (3)	(3) (3)	(2) (3)
	183	EEPPPARSGSGGGSAK		(1)	(1)
	185	EEPPPARSGSGGGSAK		(1)
	183; 185	EEPPPARSGSGGGSAK	(3)	(3)	(3)
	382	SVIGTPEFMAPEMYEEK	(1)
	599	QQVEQSSASQTGIK	(2) (3)	(3) (3)	(2)
	1220	DDYGFSGSQK	(2)	(1)
	1849	EGPVLATSSGAGVFK	(3)	(1)
	1978	EGPVASPPFMDLEQAVLPAVIPK	(3)	(3)	(3)
	2121	VPPAVIIPPAAPLSGR	(2)		(1)
	2245	GTFTDDLHK	(1)
	2372	SISNPPGSNLRTT	(3) (3)	(3) (3)	(3)
hsWNK2	560	EQQDVGSPDK	(2)	(3)	(3)
	1889	AGSLGPETPSR	(3)	(3)	(3)
	1862	QASLPVSGSVAGDFVK	(1) (3)		(2) (1)

The number in brackets indicates in how many technical replica a given phospho-site was detected (max. 3). Each bracket provides the results of one biological experiment.

Table 4

Phospho-sites of hsPKC endogenously expressed in stably transfected HEKrnKCC2b cells.

Stably transfected HEK were treated with or without 1 mM NEM or 8 μM staurosporine before they were analyzed by mass spectrometry. The protein accession numbers are: hsPKC Alpha (P17252), hsPKC Beta (P05771-2), hsPKC Delta (Q05655), hsPKC Epsilon (Q02156), hsPKC Theta (Q04759), hsPKC Eta (P24723), and hsPKC Iota (P41743).

classes	sample	Phosphorylation site	Phosphopeptides	untreated	staurosporine treated	NEM treated
conventional	PKC Alpha	10;11	ADVFPGNDSTASQDVANRFARK	(3)		(1)
		10;13	ADVFPGNDSTASQDVANRFARK	(3) (3)	(1) (1)	(1) (1)
		226	TIRSTLNPQWNESFTFK		(2)	(2)
		319	VISPSEDRK	(3) (3)	(3) (2)	(2) (3)
	PKC Beta	11	ADPAAGPPPSEGEESTVRFARK	(2)	(1)	(2)
		641	HPPVLTPPDQEVIR	(3)	(3)	(3)
novel	PKC Delta	645	ARLSYSDK	(3)	(3)	(3)
		647	ARLSYSDK	(2)	(1)	(1)
		664	NLIDSMDQSAFAGFSFVNPK	(1)		(3)
	PKC Epsilon	309	VLADLGVTPDK	(2)	(2)	(2)
	PKC Theta	685	ALINSMDQNMFR	(2)	(1)	(2)
	PKC Eta	317	TLAGMGLQPGNISPTSK	(3)	(2)	(2)
atypical	PKC Iota	564	PNISGEFGLDNFDSQFTNEPVQLTPDDDDIVRK	(3)	(1)	(3)

The number in brackets indicates in how many technical replica a given phospho-site was detected (max. 3). Each bracket provides the results of one biological experiment.

PKC mediates the phosphorylation of KCC2 and NKCC1 [48, 77, 96, 97]. The PKC family consists of 10–12 isoforms grouped into three classes [98-101]. We detected several phospho-sites in seven hsPKC family members (alpha, beta, delta, epsilon, theta, eta and iota; Table 4).

To summarize, according to mass spectrometry based phosphoproteome analyses, staurosporine and NEM reduce the number of detected phospho-sites of stably expressed rnKCC2b and endogenously expressed hsKCC1, hsKCC4, hsNKCC1, hsWNK1 and hsSPAK. Phosphorylation of some sites (rnKCC2b: Ser31, hsNKCC1:Ser242, hsKCC4: Thr980) was absent only after staurosporine treatment. Yet, these results can only be used as an indication since the absence of phosphorylation sites can reflect detection problems caused by low phosphorylation rates.

Quantitative analyses of phospho-sites of rnKCC2b and hsNKCC1 upon staurosporine and NEM treatment in HEK cells

The experimental setup of our mass spectrometry-based analysis precluded quantification of changes at individual phospho-sites. We therefore applied in a next step phospho-site-specific antibody, as they were previously shown to quantitatively monitor changes in KCC2, NKCC1 and SPAK phosphorylation [23, 48, 59, 66, 70, 102]. Currently, a limited number of this class of antibodies is available for CCCs. They are directed against the well-known phospho-sites Ser940, Thr906 and Thr1007 in rnKCC2b and Thr203, 207, 212 in hsNKCC1. So far, no data are available for the staurosporine and NEM effect on Thr906 in rnKCC2b and Thr203, 207, 212 in hsNKCC1 and the staurosporine effect on Ser940, and Thr1007 in rnKCC2b. To examine the impact of staurosporine and NEM on the phosphorylation level of these sites, we treated HEK cells with 8 μM staurosporine, 1 mM NEM or DMSO as a vehicle control for 15 min. Lysates were probed for KCC2 or NKCC1 and phosphorylation levels of each phospho-site were quantified (Fig 3). As previously described, positions Ser940, Thr906 and Thr1007 are phosphorylated in KCC2b, and Thr203, 207, 212 in NKCC1 of untreated HEK cells [48, 70, 102] (Fig 3). These data corroborate the phosphoproteome analyses which revealed phosphorylation of Ser940, Thr906 and Thr1007 in KCC2b and Thr212/Thr217 in NKCC1 as well (Table 1).

Next, we observed the impact of staurosporine and NEM on these phospho-sites (Fig 3). Both agents decreased the phosphorylation status of the WNK/SPAK sites Thr906 (p-value for NEM or staurosporine: p = 0.0026) and Thr1007 (p-value for NEM or staurosporine: p = 0.0026) of rnKCC2 and Thr203/207/212 of hsNKCC1 (p-value for NEM and staurosporine: p = 0.0026) [72–75, 103]. The reduced phosphorylation of Thr212 in hsNKCC1 agrees with our phosphoproteome analyses as no phosphorylation of Thr212/Thr217 in NKCC1 was observed (Table 1).

Previous analyses showed that SPAK directly phosphorylates Thr1007 of rnKCC2 [48] and Thr203/207/212 of hsNKCC1 [70]. Treatment of HEK293 cells with NEM resulted in a decrease of phosphorylation of Thr233 (p = 0,0026), that is located in the T-loop kinase domain, and the S-loop phosphorylation site Ser373 of hsSPAK (p = 0.0026) (Fig 3). Both are targets of WNKs [48, 69, 93]. As no data were available for the staurosporine mediated effect, we additionally analyzed its impact on these phospho-sites in HEK293 cells. Staurosporine also reduced the phosphorylation of Thr233 (p = 0.0026) and Ser373 (p = 0.0026) in hsSPAK (Fig 3). Thus, both agents reduced phosphorylation levels of these SPAK phospho-sites. These data conform well to our phosphoproteomic analyses, as no phosphorylated Ser372/373 of hsSPAK was detected after treatment with either of the two agents.

Furthermore, staurosporine reduced phosphorylation of Ser940 (p = 0.0026) in HEK, whereas NEM increased phosphorylation of Ser940 (p = 0.046) significantly (Fig 3). Since, Ser940 is directly phosphorylated by PKC [77, 104], we here analyzed the impact of both agents on the T-loop phosphorylation site Thr505 of PKC-δ. Autophosphorylation of this site is most probably essential for kinase activity [105-107]. Staurosporine significantly decreases Thr505 phosphorylation (p = 0.0026, Fig 3), whereas NEM slightly, but not significantly, increases Thr505 phosphorylation (p = 0.064). The different impact of both agents on the phosphorylation of Thr505 of PKC correlates well with their impact on Ser940 phosphorylation of KCC2b.

We also determined whether staurosporine or NEM altered the total protein amount of rnKCC2b, hsNKCC1 or hsSPAK (Fig 3). Whereas NEM resulted in increased KCC2 amount (p = 0.0026), no obvious change was detected upon staurosporine treatment. NKCC1 and SPAK levels were not changed significantly upon treatment with either agent.

To conclude, staurosporine and NEM reduced the phosphorylation status of SPAK (Thr233 and Ser373). This correlated with the reduction of the phosphorylation of Thr1007 in rnKCC2b and Thr203/207/212 in hsNKCC1. Additionally, both agents reduced phosphorylation of Thr906 in rnKCC2b, which is phosphorylated by WNKs and a yet unknown kinase [48]. Staurosporine also reduced phosphorylation of the PKC site Ser940 in rnKCC2b, whereas NEM increased its phosphorylation level. This correlated with the reduction of Thr505 phosphorylation of PKC-δ upon staurosporine treatment and the impact of NEM to increase Thr505 phosphorylation.

Quantitative analyses of phospho-sites of KCC2 and NKCC1 upon staurosporine or NEM treatment of rat immature hippocampal neurons

As KCC2 is predominantly expressed in neurons [20], we analyzed for the first time the impact of staurosporine and NEM on the phosphorylation of specific phospho-sites of endogenously expressed KCC2 and NKCC1 using immature (9 DIV) primary rat hippocampal neurons (Fig 4). At this age cultured hippocampal neurons exhibit prominent level of Thr906 and Thr1007 KCC2 phosphorylation [66] that could be a subject of modulation by staurosporine and NEM. To this end, we treated neurons with 8 μM staurosporine, 0.5 mM NEM or DMSO as a vehicle control for 15 min. NEM was reduced to 0.5 mM, since higher concentrations induced cell death. As described for the analyses in stably transfected HEK cells, we used phospho-specific KCC2 and NKCC1 antibodies to quantify phosphorylation levels of each phospho-site relatively to the DMSO control. In untreated cultured immature hippocampal neurons, Ser940, Thr906 and Thr1007 in KCC2 and Thr203, 207, 212 in NKCC1 were phosphorylated, similar to stably transfected HEK cells (Fig 4).

Most actions of staurosporine and NEM, as monitored by immunblots, were similar between HEK cells and immature hippocampal neurons. Both agents resulted in decreased phosphorylation of Thr1007 in KCC2 (p-value for NEM and staurosporine: p = 0.0026), Thr203/207/212 in NKCC1 (p-value for NEM and staurosporine: p = 0.0026), and Ser373 in SPAK (p-value for NEM: p = 0.0026 and staurosporine: p = 0.0026). Additionally, both agents reduced phosphorylation of the WNK-dependent phospho-site Thr906 in KCC2 (p-value for NEM and staurosporine: p = 0.0026). Finally, NEM increased the total protein level of KCC2, as observed in HEK293 cells (p = 0.015) (Fig 4). We were not able to detect the phosphorylation of Thr233 of SPAK using phospho-specific antibodies.

A marked difference, however, was observed for the PKC dependent phospho-site Ser940. Here, treatment with NEM reduced phosphorylation of Ser940 (p = 0.0026) (Fig 4), contrary to the results obtained in stably transfected HEK cells (Fig 3). Treatment with staurosporine also resulted in reduced phosphorylation of Ser940 (p = 0.0026), which was similar to its action in HEK cells. We could not detect Thr505 phosphorylation of PKC-δ due to low expression rates.

To sum up, NEM affected the phosphorylation status of Ser940 in immature hippocampal neurons (decrease) in the opposite way compared to HEK cells (increase). All other effects of staurosporine and NEM were similar between immature hippocampal neurons and HEK293 cells, i.e. both reduced the phosphorylation status of Ser940, Thr906, and Thr1007 in KCC2 and Thr203/207/212 in hsNKCC1.

Discussion

KCC2 and NKCC1 are key players for the development and maintenance of fast inhibitory neurotransmission. Their oppositely directed transport of K+ and Cl- ions within the same neuronal population necessitates a precisely coordinated regulatory mechanism for efficient setting of the intracellular [Cl-] concentration [4, 38, 55–60] (Fig 5).

Fig 5

Staurosporine and NEM impair the WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

In immature hippocampal neurons, the WNK mediated phosphorylation of SPAK/OSR1 directly phosphorylates T1007 of KCC2 and T203, T207 and T212 of NKCC1. WNKs also interact with a yet unknown kinase to phosphorylate Thr906 of KCC2. Phosphorylation of these residues decreases KCC2 and increases NKCC1 activity. Application of staurosporine or NEM decreases phosphorylation of S382 of WNK1 and T233 and S373 of SPAK. This likely results in reduction of phosphorylation of T906 and T1007 in KCC2 and of T203, T207 and T212 in NKCC1. Dephosphorylation of these residues increases KCC2 activity and decreases NKCC1 activity. The figure is modified from Moore et al. [39].

Posttranslational regulation via the WNK-SPAK/OSR1 dependent phosphorylation represents a potent mechanism to regulate transport activity of KCC2 and NKCC1 in a reciprocal way [60, 108–110] (Fig 5). Here, we show that staurosporine and NEM decrease phosphorylation of Thr233 and Ser373 in SPAK, of Thr1007 in rnKCC2 and Thr203, Thr207 and Thr212 in hsNKCC1 in both HEK293 cells and immature cultured hippocampal neurons. Since SPAK directly impairs phosphorylation of Thr1007 in rnKCC2 and Thr203, Thr207 and Thr212 in hsNKCC1 [38, 48, 59, 60, 63, 68–76], our data suggest that staurosporine and NEM directly affect the WNK-SPAK/OSR1 mediated phosphorylation of these residues in KCC2 and NKCC1. The data are in line with previous analyses showing that NEM reduces phosphorylation of Ser373 in SPAK and Thr1007 in KCC2 using HEK293 cells and immature cortical neurons [48]. Furthermore, application of staurosporine and NEM decreases phosphorylation of Thr906 in rnKCC2. This site is directly phosphorylated by WNKs and a yet unknown kinase [48]. However, functional in-depth analyses such as mutagenic approaches are required to prove a causal relation between dephosphorylation of SPAK Thr233 and Ser373 and dephosphorylation of the specific KCC2 and NKCC1 phospho-sites upon staurosporine and NEM treatment.

Recent analyses demonstrated that dephosphorylation of Thr906 and Thr1007 increases KCC2 activity [61, 64–66], whereas dephosphorylation of Thr203, Thr207 and Thr212 decreases NKCC1 activity [45, 51]. Furthermore, staurosporine and NEM results in activation of KCC2 (this study and [40, 47, 48, 59, 60, 79, 80], whereas they reduce NKCC1 activity [43–46, 51]. This suggests, that staurosporine and NEM mediated dephosphorylation of these phospho-sites result in a reciprocal regulation of KCC2 (activation) and NKCC1 (inactivation) activity most likely via the WNK/SPAK-dependent phosphorylation pathway. This is also in line with the observation in immature hippocampal neurons that KCC2 can rapidly be activated by staurosporine [42].

Another key regulatory KCC2 phospho-site is the PKC-mediated phosphorylation of Ser940. Phosphorylation of Ser940 enhances KCC2 cell surface expression and increases ion transport activity, whereas mutation of serine to alanine (mimicking the dephosphorylated state) results in transport activity that is equal or decreased compared to wild-type KCC2 (KCC2wt) [65, 77, 111]. Our data demonstrate that staurosporine and NEM can differentially affect Ser940 phosphorylation. Treatment of immature hippocampal neurons with either agent results in decreased phosphorylation of Ser940. However, treatment of HEK cells with staurosporine decreases, whereas NEM increases phosphorylation of Ser940. This is in line with the different effect of these agents on transport activity of the phosphomutants Ser31A/D, Thr34A/D, Thr999A and Thr1008A/D [40].

Moreover, NEM has a cell-type specific impact on Ser940 phosphorylation. In immature cortical neurons [48] and HEK293 cells, NEM increases Ser940 phosphorylation, whereas it decreases Ser940 phosphorylation in immature cultured hippocampal neurons (Fig 4). The mechanisms that cause this opposite effect of NEM on Ser940 phosphorylation in different tissues is unclear. One possibility is that different NEM concentrations (HEK293 cells: 1 mM and 0.1 mM; immature hippocampal neurons: 0.5 mM; immature cortical neurons: 0.1 mM [48]) affect different regulatory pathways. Alternatively, tissues-specific sets of PKC isoforms and phosphatases result in different phosphorylation patterns. Indeed, the PKC family consists of 10–12 isoforms grouped into three classes [98-101] that vary in their expression profile [112] and regulation of their activity through several regulatory proteins, co-factors and second messenger cascades [100, 101, 113]. This offers the opportunity to differentially regulate KCC2 function in distinct neuronal populations through PKC. In HEK cells, we showed that staurosporine reduced and NEM slightly increased phosphorylation of the T-loop phospho-site Thr505 of PKC-δ. This correlated with decreased KCC2 Ser940 phosphorylation upon staurosporine treatment and increased phosphorylation of this residue upon NEM treatment. This suggests, that both agents directly act on PKC-δ mediating the phosphorylation of Ser940 in HEK cells. However, more functional in-depth analyses are required to elucidate the causal link between dephosphorylation of PKC-δ Thr505 and dephosphorylation of KCC2 Ser940 upon staurosporine treatment. Since we were unable to detect phosphorylation of PKC-δ Thr505 in immature hippocampal neurons, we suggest that other PKC isoforms are involved in the direct phosphorylation of KCC2 Ser940 in immature hippocampal neurons.

Our data furthermore reveal that staurosporine (HEK cells, immature hippocampal neurons) and NEM treatment (immature hippocampal neurons) decrease Ser940 phosphorylation resulting in an equal or diminished transport activity compared to KCC2wt [65, 77, 111]. We therefore conclude that Ser940 is not the key regulatory phospho-site mediating the staurosporine and NEM-based stimulation effect on KCC2. This is in line with recent published analyses showing that NEM still enhances the transport activity of Ser940A (mimicking dephosphorylated state), indicating that other phospho-sites are important in NEM-dependent stimulation [40, 48].

NEM but not staurosporine increased total KCC2 amount in HEK293 cells and immature cultured hippocampal neurons. In immature cortical neurons, Deep and coworkers [48] detected the same trend of enhanced total KCC2 abundance (albeit not significant), resulting in increased cell surface expression [48]. This suggests that in contrast to staurosporine, NEM increases KCC2 expression and trafficking that could result in a higher KCC2 activity. The different impact of NEM on total KCC2 abundance in HEK293 in the study of Deep and coworkers (no increase) and our analyses (increase) could result from different NEM concentration used in the experiments (0.1 mM vs. 1 mM).

Via mass spectrometry analysis, we identified several new phosphorylation sites whose function awaits further investigation. These sites are the N-terminal KCC2 phospho-site Thr32, and the C-terminal NKCC1 phospho-sites Ser242, Thr266, Th268, Ser940, Tyr956 and Ser957. Future studies should investigate their regulatory impact on KCC2 and NKCC1 activity.

Conclusions

In conclusion, our data identify molecular mechanisms involved in staurosporine and NEM mediated changes in transport activity of KCC2 and NKCC1, which are a defining feature of CCCs [114]. The observation of cell-type specific action of these agents is in line with different reversal potentials in mature neuronal populations [115] and calls for comprehensive neuron subtype-specific phospho analysis. The recently reported structural data of CCCs [116-119] finally lay the foundation to analyze jointly the physiological role of phosphorylation and underlying structural changes to obtain an integrative and mechanistic view of the action of phosphorylation.

Phospho-sites in PhosphoSitePlus detected by mass spectrometry analyses.

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Quantitative analyses of rnKCC2 and hsNKCC1 phospho-sites upon staurosporine and NEM treatment in HEKrnKCC2b cells.

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Quantitative analyses of rnKCC2 and hsNKCC1 phospho-sites upon staurosporine and NEM treatment in immature hippocampal neurons.

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